1. Field of the Invention
The present invention relates to a method of plant transformation in which a DNA construct carrying a gene encoding the green fluorescent protein (GFP) is introduced into plant cells which are then screened for the presence of the protein and transformed cells are regenerated into transgenic plants. In particular, the present invention provides methods for circumventing the cellular toxicity of the GFP by regulating expression of the gene encoding the protein or directing the protein to a subcellular compartment where it is not toxic to the cell. The present invention provides DNA constructs for cell transformation in which expression of a gene encoding the GFP is placed under the control of an inducible, constitutive or tissue-specific promoter. In addition, DNA constructs are provided in which a nucleotide sequence encoding the GFP is operably linked to a signal or targeting sequence which directs the expressed protein to a subcellular compartment where the protein is not toxic to the cell. Moreover, the present invention provides a nucleotide sequence encoding GFP that is optimized for expression of the GFP gene in plants and to GFP-encoding nucleotide sequences that code for light-shifted versions of GFP. The present invention also provides a method for selecting plant cells transformed with a gene encoding a screenable marker flanked on the 5-prime and 3-prime ends with a recombinase-specific target sequence, and introducing a gene encoding a site specific recombinase into the transformed plant cells and selecting transformed plant cells that no longer express the screenable marker. In addition, the present invention provides a method of reducing GFP toxicity by transforming plant cells with a gene encoding the GFP together with a gene encoding an oxygen scavenger such as superoxidase dismutase.
2. Background
Expression vectors include at least one genetic marker that allows transformed cells to be either recovered by negative selection, i.e. inhibiting growth of cells that do not contain the selectable marker gene, or by screening for product encoded by the genetic marker. Many of the commonly used selectable marker genes for plant transformation were isolated from bacteria and code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or a herbicide. Other selectable marker genes encode an altered target which is insensitive to the inhibitor.
The most commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase II (nptII) gene, isolated from Tn5, which when placed under the control of plant regulatory signals confers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80: 4803 (1983). Another commonly used selectable marker gene is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5: 299 (1985).
Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3xe2x80x2-adenyl transferase, the bleomycin resistance determinant. Hayford et al., Plant Physiol. 86: 1216 (1988), Jones et al., Mol. Gen. Genet., 210: 86 (1987), Svab et al., Plant Mol. Biol. 14: 197 (1990), Hille et al., Plant Mol. Biol. 7: 171 (1986). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or broxynil. Comai et al., Nature 317: 741-744 (1985), Gordon-Kamm et al., Plant Cell 2: 603-618 (1990) and Stalker et al., Science 242: 419-423 (1988).
Other selectable marker genes for plant transformation are not of bacterial origin. These genes include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13: 67 (1987), Shah et al., Science 233: 478 (1986), Charest et al., Plant Cell Rep. 8: 643 (1990).
Although many of these markers have been used for selecting transformed plant tissue, these selection systems involving toxic chemical agents can have disadvantages or limitations. One disadvantage is that it may be difficult to recover normal, viable transformed plants directly from chemical selection. Everett et al., Bio/Technology 5: 1201-1204 (1987). Another disadvantage is that not all selectable marker systems work for all tissues, in all plant species, due in part to differences in sensitivity of a particular tissue or plant species to the selective agent. The success of any given marker for transformation of a given plant species is not easily predicted. Moreover, potential regulatory issues surrounding the use of antibiotic resistance genes and the use of herbicide resistance genes for plant species capable of outcrossing with weedy species are additional disadvantages of these markers.
Another class of marker genes for plant transformation require screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantify or visualize the spatial pattern of expression of a gene in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening presumptively transformed cells include xcex2-glucuronidase (GUS), xcex2-galactosidase, luciferase, and chloramphenicol acetyltransferase Jefferson, R. A., Plant Mol. Biol. Rep. 5: 387 (1987)., Teeri et al., EMBO J. 8: 343 (1989), Koncz et al., Proc. Natl. Acad. Sci. U.S.A. 84: 131 (1987), De Block et al., EMBO J. 3: 1681 (1984). Another approach to the identification of relatively rare transformation events has been use of a gene that encodes a dominant constitutive regulator of the Zea mays anthocyanin pigmentation pathway. Ludwig et al., Science 247: 449 (1990).
Although chemical selection of plant cells transformed with selectable marker genes has been successful with plant species and varieties that are easily cultured in vitro, the choice of selectable marker systems that have been shown to be successful for cereals and many other agronomically important plant species is very limited. In general, plant species that tend toward organogenesis and/or shoot propagation have been difficult to transform by means of chemical selection. The success rate with these plant species continues to improve, however, as evidenced by recent advances in Type I selection of maize inbreds and small grain cereals such as barley. Koziel et al., Bio/Technology 11: 194-200 (1993) and Mendel et al. In: Transformation of Plants and Soil Microorganisms, Wang et al. eds., Cambridge Press (1995).
Likewise, there has been little success in using visual screening methods for primary identification of transformed cells. The GUS gene was used to investigate germline transmission. McCabe et al., Plant Physiol., 87(3): 671 (1988) and McCabe et al., Plant Cell Tissue Organ Cult. 33 (3): 227 (1993). Histochemical staining for GUS activity was used to locate transgenic sectors in cotton and soybean transformants that ultimately produced transgenic seeds. Since histochemical analysis for GUS activity requires destruction of portions of the presumptively transformed plant tissue, this method is labor intensive and impractical for routine production of transgenic plants. This method is particularly unsuitable for plant species such as maize and other cereals in which transformants are recovered, even under optimum conditions, at low frequency. Recovery of transformed progeny was reported once in barley using GUS expression as a screening tool, but the method was found to be very labor-intensive. Ritala et al., Plant Mol. Biol. 24: 317-325 (1994). There have been no reports at all of success with GUS or other screenable markers with maize.
More recently, in vivo methods for visualizing GUS activity that do not require destruction of plant tissue have been made available. Molecular Probes Publication 2908, Imagene Green(trademark), p. 1-4 (1993) and Naleway et al., J. Cell Biol. 115: 151a (1991). However, these in vivo methods for visualizing GUS activity have not proven useful for recovery of transformed cells because of low sensitivity and high fluorescent backgrounds.
Despite the fact that luciferase genes have been available for many years, this strategy for visualizing transformed cells has also not been successfully adapted for routine recovery of plant transformants. The luciferase-based screening methods are limited by the fact that most of these systems require the presence of luciferin, a compatible luciferase and an exogenously supplied cofactor. In the absence of the substrate, enzyme or cofactor, the system does not bioluminesce. Cells transformed with a luciferase gene must have cell walls and plasma membranes that are permeable, or rendered permeable, to a compatible luciferin in order to detect bioluminescence. For example, tobacco plants regenerated from cells transformed with a firefly luciferase gene and exposed to a liquid medium containing firefly luciferin exhibited bioluminescence primarily along their major veins. Ow et al., Science 234: 856 (1986). Accordingly, a screening method has not been successfully developed for routine plant transformation that does not involve chemical selection or assays, often labor-intensive, that require the sacrifice or destruction of tissue samples for analysis.
A gene encoding GFP has been utilized as a marker for gene expression in prokaryotic and eukaryotic cells. Chalfie et al., Science 263: 802 (1994). Many cnidarians utilize GFPs as energy-transfer acceptors in bioluminescence. A gene encoding GFP isolated from a cnidarian and expressed in a heterologous prokaryotic or eukaryotic host produces a protein capable of fluorescence. A cDNA encoding the Aequorea victoria GFP produced a fluorescent product when expressed in Escherichia coli or Caenorhabditis elegans cells. Green fluorescence was detected in transformed cells upon illumination with a long-wave ultraviolet (UV) source without having to supply substrates or cofactors. In addition, fluorescence was stable for at least 10 min when illuminated with 450 to 490 nm light. Transformation of plant cells with a gene encoding GFP and detection of fluorescence has been reported. Haseloff et al., TIG 11: 328-329 (1995). Transformed Arabidopsis cells could be regenerated into whole plants. However, the regenerated plants expressing GFP exhibited signs of mild to moderate toxicity in the light compared to plants not expressing GFP. The strongest GFP expressors proved more difficult to regenerate. Likewise, in a recent report describing GFP expression in kanamycin-selected tobacco transformants, Chiu et al., Current Biol. 6: 325-330 (1996) note that high GFP expression levels inhibited regeneration of transgenic plants.
A need therefore exists for a cell transformation method which does not rely, or does not rely solely on, selection of cells carrying a gene that confers resistance to a toxic substance. A need exists for a method for efficiently and easily identifying transformed plant cells with a visual screenable marker. A need also exists for a method of cell transformation that does not require destruction of presumptively transformed tissue to assay for the presence of a selectable marker gene. A need exists for a method for cell transformation that combines a selectable marker gene and a screenable marker gene. In addition, a need exists for a method of cell transformation which does not require exogenous supply of a substrate or cofactor for detection of the polypeptide encoded by a selectable marker gene. Yet another need exists for a method of cell transformation that circumvents the cellular toxicity of the GFP.
Accordingly, it is an object of the present invention to provide a method for cell transformation which does not depend on selection of cells carrying a gene that confers resistance to a toxic substance.
It is another object of the present invention to provide a method for cell transformation which combines the selection of cells carrying a gene that confers resistance to a toxic substance with screening cells for the presence of a substance that renders transformed cells identifiable.
It is a further object of the present invention to provide a method for cell transformation which does not require destruction of presumptively transformed tissue to assay for the presence of a selectable marker gene.
Yet another object of the present invention is to provide a method for cell transformation which does not require that an exogenous substrate or cofactor be provided to assay for a polypeptide encoded by a selectable marker gene.
It is another object of the present invention to provide a method for cell transformation that circumvents the cellular toxicity of the GFP.
These and other objects are achieved, in accordance with one embodiment of the present invention, by providing an isolated DNA molecule comprising a nucleotide sequence encoding the GFP operably linked to an inducible promoter. The inducible promoter can be selected from the group consisting of the estrogen-inducible promoter, the estradiol-inducible promoter, the ACE1 promoter, the IN2 promoter and the tetracycline repressor promoter.
Also provided is an isolated DNA molecule comprising as nucleotide sequence encoding the GFP wherein the nucleotide sequence is operably linked to a targeting sequence for subcellular localization which directs a protein to a subcellular compartment.
An isolated DNA molecule is provided comprising a nucleotide sequence selected from the group consisting of (a) SEQ ID NO: 1; (b) a nucleotide sequence that has substantial sequence similarity with SEQ ID NO: 1; and (c) a functional fragment of (a) or (b), wherein said DNA molecule encodes a GFP. The nucleotide sequence encoding a GFP may be operably linked to a nucleotide sequence encoding a targeting sequence for subcellular localization which directs a protein to a subcellular compartment. The nucleotide sequence encoding a GFP and a targeting sequence may further comprise a promoter operably linked to said nucleotide sequence.
Also provided are expression vectors comprising DNA molecules encoding a GFP, optionally operably linked to an inducible promoter and/or targeting sequence. The expression vector of the instant invention may carry a nucleotide sequence encoding a foreign protein operably linked to a second promoter.
Also provided is a method of using the expression vectors of the instant invention to produce a transformed plant, comprising the steps of introducing an expression carrying a gene encoding GFP into regenerable plant cells and selecting cells containing the GFP for regeneration. The method of the instant invention may include the step of inducing GFP expression where the nucleotide sequence encoding GFP is operably linked to an inducible promoter. The regenerable plant cells utilized in the method of the instant invention are selected from the group consisting of Zea, Brassica and Helianthus cells.
Also provided are transgenic plants expressing the isolated DNA molecule encoding GFP. In addition, transgenic plants comprising a vector carrying a nucleotide sequence encoding GFP are provided.
A method for producing a transgenic plant is provided comprising the steps of (a) constructing an expression vector comprising (i) a first promoter which is an inducible promoter, operably linked to a nucleotide sequence encoding a GFP, and (ii) a second promoter operably linked to a foreign gene; (b) introducing said expression vector into regenerable plant cells; (c) inducing expression of the gene encoding the GFP and selecting transformed plant cells containing said protein; and (d) regenerating transformed plants from said selected transformed plant cells. The inducible promoter may be selected from the group including the estrogen-inducible promoter, the estradiol-inducible promoter, the ACEI promoter, the IN2 promoter and the tetracycline repressor promoter.
Also provided is a method for producing a transgenic plant, comprising the steps: (a) constructing an expression vector comprising (i) a first promoter operably linked to a nucleotide sequence encoding a sequence for subcellular localization which directs a protein to a subcellular compartment which is operably linked to a nucleotide sequence encoding a GFP, and (ii) a second promoter operably linked to a foreign gene; (b) introducing said expression vector into regenerable plant cells; (c) selecting transformed plant cells containing said GFP; and (d) regenerating transformed plants from said selected transformed plant cells. The targeting sequence for subcellular localization directs the GFP to the mitochondria, chloroplasts, peroxisomes, vacuole, endoplasmic reticulum. cell wall or for secretion generally into the apoplast. This method may include a nucleotide sequence encoding a GFP selected from the group consisting of: (a) SEQ ID NO: 1; (b) a nucleotide sequence that has substantial sequence similarity with SEQ ID NO: 1; and (c) a functional fragment of (a) or (b).
Also provided is a method for producing a transgenic plant, comprising the steps: (a) constructing an expression vector comprising (i) a first promoter operably linked to a nucleotide sequence encoding a screenable marker flanked on the 5-prime and 3-prime ends with a recombinase-specific target sequence, and (ii) a second promoter operably linked to a foreign gene; (b) introducing said expression vector into regenerable plant cells; (c) selecting transformed plant cells containing said screenable marker; (d) transforming the plant cells with a second expression vector containing a gene encoding a site-specific recombinase; (e) selecting plant cells that no longer express the screenable marker; (f) regenerating transformed plants from said selected transformed plant cells; and (g) isolating said foreign protein. The site-specific recombinase may be selected from the group consisting of the FLPtFRT, Ac/DS and cre/lox systems. In addition, the screenable marker may be the GFP.
An isolated DNA molecule comprising a nucleotide sequence encoding the GFP fused in frame to a nucleotide sequence encoding superoxide dismutase is also provided. Cells transformed with the DNA molecule contain a fusion protein that (i) produces green fluorescence in the presence of UV to blue light and (ii) displays superoxide dismutase activity.
Also provided is a method for producing a transgenic plant comprising: (a) constructing an expression vector comprising (i) a first promoter operably linked to a nucleotide sequence encoding a GFP, and (ii) a second promoter operably linked to a gene encoding an enzyme that is an oxygen scavenger, and (iii) a third promoter operably linked to a foreign gene; (b) introducing said expression vector into regenerable plant cells; (c) selecting transformed plant cells containing said GFP; and (d) regenerating transformed plants from said selected transformed plant cells. The oxygen scavenger enzyme may be superoxide dismutase.
Alternatively, a method for producing a transgenic plant is provided comprising: (a) constructing an expression vector comprising (i) a first promoter operably linked to a nucleotide sequence encoding a fusion protein comprising the GFPand an oxygen scavenger enzyme fused in frame, and (ii) a second promoter operably linked to a foreign gene; (b) introducing said expression vector into regenerable plant cells; (c) selecting transformed plant cells containing said the GFP and oxygen scavenger enzyme activity; and (d) regenerating transformed plants from said selected transformed plant cells. The oxygen scavenger enzyme may be superoxide dismutase.